Systems and methods for delivering nucleic acids to a plant

ABSTRACT

A method of delivering at least one nucleic acid to a plant, comprising: applying a light energy to a surface of the plant to create a pore; and applying at least one nucleic acid to the pore, wherein the at least one nucleic acid comprises RNA, wherein the at least one nucleic acid comprises at least one of a RNA interference (RNAi), an antisense RNA (asRNA), a micro RNA (miRNA), a small interfering RNA (siRNA), a double-stranded RNA (dsRNA), a non-coding RNA (ncRNA), mitochondrial RNA (mtDNA), and combinations thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/313,634 filed on Mar. 25, 2016, entitled “Systems and Methods forDelivering Nucleic Acids to a Plant”, the contents of which areincorporated herein by reference as though set forth in their entirety.

BACKGROUND

This invention is directed to devices and methods for delivering nucleicacids into a plant using a laser.

The background description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Plant pathogens pose numerous challenges to the plant industry. One ofthe most serious challenges involves controlling pathogens to preventsignificant plant losses. For example, Huanglongbing (hereinafterreferred to as “HLB”) or citrus greening disease has led to a loss ofmany citrus trees. Consequently, HLB may be very damaging to the cropyield in large scale operations.

Some have contemplated using RNA interference (hereinafter referred toas “RNAi”) to improve the characteristics of a plant. For example,Younis et al., contemplates RNAi as a tool to engineer pathogenresistant plants, insect/pest resistant plants, and improved qualityplants. Adnan Younis et al., RNA Interference (RNAi) Induced GeneSilencing: A Promising Approach of Hi-Tech Plant Breeding, InternationalJournal of Biological Sciences 2014, Vol. 10: 1150-1158. Others havecontemplated similar uses of RNAi in plants, including Karl H. J. Gordonet al., RNAi for insect-proof plants, Nature Biotechnology 2007, Vol.10: 11, Hajeri et al., Citrus tristeza virus-based RNAi in citrus plantsinduces gene silencing in Diaphorina citri, a phloem-sap sucking insectvector of citrus greening disease (Huanglongbing), Journal ofBiotechnology 176 (2014) 42-49, and Wie Tang et al., Efficient deliveryof small interfering RNA to plant cells by a nanosecond pulsedlaser-induced stress wave for posttranscriptional gene silencing, PlantScience 171 (2006) 375-381. These and all other extrinsic materialsdiscussed herein are incorporated by reference in their entirety. Wherea definition or use of a term in an incorporated reference isinconsistent or contrary to the definition of that term provided herein,the definition of that term provided herein applies and the definitionof that term in the reference does not apply.

Nucleic acids, and more specifically RNAi, may be used to enhance aplant as briefly described above. Some have contemplated delivering RNAinto a plant to control pathogen and/or pest control damage. Forexample, Tang (US 2013/0047298) contemplates spraying or brushing thesurface of a plant with a double-stranded RNA (hereinafter referred toas “dsRNA”) to promote penetration of dsRNA into plant cells. Althoughspraying or brushing is less invasive than other delivery methods, itappears that the uptake of RNA by spraying or brushing may becompromised due to the relatively low permeability of the outer layer ofsome plants.

Thus, there is still a need in the art for improving uptake of nucleicacids (e.g., RNAi) in plants.

SUMMARY

The following presents a simplified overview of the example embodimentsin order to provide a basic understanding of some embodiments of theexample embodiments. This overview is not an extensive overview of theexample embodiments. It is intended to neither identify key or criticalelements of the example embodiments nor delineate the scope of theappended claims. Its sole purpose is to present some concepts of theexample embodiments in a simplified form as a prelude to the moredetailed description that is presented later. It is to be understoodthat both the following general description and the following detaileddescription are exemplary and explanatory only and are not restrictive.

In accordance with the embodiments disclosed herein, the presentdisclosure is directed to an apparatus, systems, and methods in whichlight energy may be used to enhance uptake of nucleic acids in plants.As used herein, the term “plant” means any type of plant life, includinga tree, vines, forage, perennial crops, row crops, bush crops, anornamental plant, annual plants, and grasses. A light energy may beapplied to a surface of a plant to create a pore, and the nucleic acidis applied to the pore. It should be appreciated that a pore may be arupture, an ablation, a disruption or a minor incision. Thus, geneticactivity within the plant may be efficiently and effectively modified.

In one embodiment, a plant having a disease may be treated. For example,light energy may be applied to a treatment area to create a pore, andnucleic acid may be applied to the pore in an amount effective to treata disease in an infected site. Contemplated diseases are typicallysystemic in nature, which means that a pathogen occurs within planttissue. However, it is also contemplated that surface diseases (e.g.,citrus canker) may also be treated. Thus, instead of applying excessivedosages of a nucleic acid via a spray to treat surface diseases, it iscontemplated that the dosage amount of the nucleic acid may be greatlyreduced by feeding the nucleic acid to a pore created by the lightenergy. It should be appreciated that treating a plant does not requirea complete cure of the plant from the disease, but may also include areduction in the disease state or a reduction in a symptom of thedisease.

In one embodiment, the step of applying a light energy includes creatinga pore pattern. The pore pattern may be a single pore or a plurality ofpores, wherein the plurality of pores may be arranged in a single line,a plurality of lines, a continuously bending line (e.g., swirls, randomcurves, etc.), a plurality of continuous bending lines, and combinationsthereof. It is contemplated that the pore pattern may influence theamount of the first dosage of the nucleic acid that may be required tobe effective to induce a therapeutic response. Furthermore, the firstpore may have a diameter of approximately 100 to 300 μm. It should beappreciated that the various pore depths, widths, lengths, surfaceareas, and patterns may be used to promote a therapeutic response in aminimally invasive manner. It should be understood that the terms“pore,” “first pore,” “second pore”, or the like may refer to singlepore or a plurality or grouping of pores.

In one embodiment, the step of applying a light energy may be before thestep of applying the first dosage. However, it should be appreciatedthat the reverse order may also be performed to treat plant. Apredetermined time may also be used to regulate the performance of suchsteps to ensure effective uptake of the nucleic acid into the plant viathe first indentation. For example, the step of applying a light energyand the step of applying a first dosage may be performed in less than anhour, less than 30 minutes, or even less than 1 minute from one another.

As briefly mentioned above, the infected site may be in a phloem. Insuch embodiment, suitable treatment areas include a surface of at leastone of a leaf, a stem, and a bark of the plant. For example, a plant canbe a citrus tree having HLB. The citrus tree having HLB has an infectedsite of the phloem. To treat the citrus tree, light energy can beapplied to create a first pore or pores on a treatment area, which caninclude at least one of a leaf, a steam and a bark. Once the pore iscreated, a first dosage of a nucleic acid may be applied to thetreatment area in an amount effective to induce a therapeutic response(e.g., reduce a symptom of HLB). It should be appreciated that the firstpore may comprise a rupture of a cuticle on the leaf to allowintroduction of the nucleic acid.

While the embodiments described above have discussed a light energycreating a first pore, it is contemplated that the step of applying thelight energy to the treatment area may include creating a second pore.With a second pore, the first dosage of the nucleic acid may bebeneficially received by the first and second pores. Thus, additionalpores may be contemplated so long as the amount of pores created isminimally invasive to prevent or reduce damage to the plant.

In order to reduce the risk of damage to the plant, in one embodiment,wax may be applied to the treatment area in an amount effective to sealthe first pore after the steps of applying the light energy to thetreatment area and applying the first dosage of a nucleic acid to thetreatment area are complete. As a further step of precaution anantifungal spray may be applied to the treatment area.

The treatment area and the infected site may be in distinct plantsystems within the plant. For example, the treatment area may be in thedermal system of the plant (e.g., cuticle, cork) while the infected sitemay be in the vascular system of the plant (e.g., phloem). In addition,as briefly discussed above, the treatment area and infected site may bedistal. As used herein, “distal” means situated away from. In someinstances, distal may refer to a physical distance between the treatmentarea and the infected area, for example, a distance of between 0.01 cmto 100 cm, more preferably, between 0.05 cm to 50 cm, and mostpreferably, between 0.1 cm to 25 cm. In other instances, distal mayrefer to a distance between treatment area and infected site in terms ofa systemic pathway. For example, the treatment area and the infectedsite are distal by one to two layers of a plant epidermis or one to twolayers of bark tissues. In addition, distal could refer to a systemicdistance that crosses multiple systems (e.g., dermal system, vascularsystem, ground system). Moreover, distal could refer to locations ondifferent branches and/or leaves of a plant.

In another embodiment, a first nucleic acid may be delivered to a firstsite in a plant. In such embodiment, a light energy may be applied to asecond site located on a plant surface. The first site (e.g., phloem,xylem) and the second site (e.g., cuticle, cork) may be different inkind. In other words, the first site and the second site may bedifferent plant systems (e.g., dermal system, vascular system) ordifferent plant tissues. A first dosage of the first nucleic acid may beapplied to the second site in an amount effective to promote uptake ofthe first nucleic acid and delivery to the first site via the secondpore.

The plant may include a tree, row crop, a bush crop, and an ornamentalplant. Furthermore, as a precautionary measure, a wax may be applied tothe second site in an amount effective to seal the second pore after thesteps of applying the light energy to the second site and applying thefirst dosage of the nucleic acid to the second site.

Still other advantages, embodiments, and features of the subjectdisclosure will become readily apparent to those of ordinary skill inthe art from the following description wherein there is shown anddescribed a preferred embodiment of the present disclosure, simply byway of illustration of one of the best modes best suited to carry outthe subject disclosure As it will be realized, the present disclosure iscapable of other different embodiments and its several details arecapable of modifications in various obvious embodiments all withoutdeparting from, or limiting, the scope herein. Accordingly, the drawingsand descriptions will be regarded as illustrative in nature and not asrestrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are of illustrative embodiments. They do not illustrate allembodiments. Other embodiments may be used in addition or instead.Details which may be apparent or unnecessary may be omitted to savespace or for more effective illustration. Some embodiments may bepracticed with additional components or steps and/or without all of thecomponents or steps which are illustrated. When the same numeral appearsin different drawings, it refers to the same or like components orsteps.

FIG. 1 is a flow diagram of a method of treating a plant having adisease in an infected site of the plant.

FIGS. 2A-2F are perspective views of an embodiment showing deliver ofthe first nucleic acid from a treatment area to an infected site.

FIGS. 3A-3F are perspective views of an embodiment showing deliver of afirst nucleic acid from a treatment area to an infected site separatedby a systemic pathway.

FIGS. 4A-4D illustrates various embodiments of pore patterns.

FIGS. 5A-5B is a top and perspective view of embodiments for deliveringa nucleic acid to a plant.

DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

Before the present methods and systems are disclosed and described, itis to be understood that the methods and systems are not limited tospecific methods, specific components, or to particular implementations.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting.

As used in the specification and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Ranges may be expressed herein as from “about” oneparticular value, and/or to “about” another particular value. When sucha range is expressed, another embodiment includes from the oneparticular value and/or to the other particular value. Similarly, whenvalues are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms anotherembodiment. It will be further understood that the endpoints of each ofthe ranges are significant both in relation to the other endpoint, andindependently of the other endpoint.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other components, integers or steps.“Exemplary” means “an example of” and is not intended to convey anindication of a preferred or ideal embodiment. “Such as” is not used ina restrictive sense, but for explanatory purposes.

Disclosed are components that may be used to perform the disclosedmethods and systems. These and other components are disclosed herein,and it is understood that when combinations, subsets, interactions,groups, etc. of these components are disclosed that while specificreference of each various individual and collective combinations andpermutation of these may not be explicitly disclosed, each isspecifically contemplated and described herein, for all methods andsystems. This applies to all embodiments of this application including,but not limited to, steps in disclosed methods. Thus, if there are avariety of additional steps that may be performed it is understood thateach of these additional steps may be performed with any specificembodiment or combination of embodiments of the disclosed methods.

In the following description, certain terminology is used to describecertain features of one or more embodiments. For purposes of thespecification, unless otherwise specified, the term “substantially”refers to the complete or nearly complete extent or degree of an action,characteristic, property, state, structure, item, or result. The exactallowable degree of deviation from absolute completeness may in somecases depend on the specific context. However, generally speaking, thenearness of completion will be so as to have the same overall result asif absolute and total completion were obtained. The use of“substantially” is also equally applicable when used in a negativeconnotation to refer to the complete or near complete lack of an action,characteristic, property, state, structure, item, or result.

As used herein, the terms “approximately” and “about” generally refer toa deviance of within 5% of the indicated number or range of numbers. Inone embodiment, the term “approximately” and “about”, may refer to adeviance of between 0.01-10% from the indicated number or range ofnumbers.

Various embodiments are now described with reference to the drawings. Inthe following description, for purposes of explanation, numerousspecific details are set forth in order to provide a thoroughunderstanding of one or more embodiments. It may be evident, however,that the various embodiments may be practiced without these specificdetails. In other instances, well-known structures and devices are shownin block diagram form in order to facilitate describing theseembodiments.

It has been discovered that light energy may be used to enhance thepenetration of a nucleic acid in plants. This may be accomplished byapplying a light energy to a first portion of a plant to create anindentation. Once the indentation is created, a first dosage of asubstance may be applied to the plant. It should be appreciated that atleast a portion of the first dosage may penetrate the plant via theindentation.

FIG. 1 shows a schematic of a method 100 for treating a plant having aninfected site. The method 100 may comprises a step 102 of applying alight energy to a treatment area on the plant to create a first pore.Typically, the treatment area may be distal from the infected site. Inanother step 104, a first dosage of a nucleic acid may be applied to thetreatment area in an amount effective to induce a therapeutic responsein the infected site. Suitable therapeutic responses may include, butare not limited to, a complete cure of a disease causing the infectedarea, a reduction in the infected area, a reduction in a symptom of thedisease causing the infected area, a health stimulant, and combinationsthereof.

In preferred embodiments, step 102 of applying the light energy to thetreatment area may occur prior to step 104 of applying the first dosageof the nucleic acid. However, it is contemplated that the reverse ordermay be performed to treat a plant whereby step 104 occurs prior to step102. Regardless of the order in steps 102 and 104, a predetermined timemay be used to determine when each step may be completed with respect toone another. For example, step 102 and step 104 may be completed withinless than 1 hour, more preferably less than 30 minutes, and mostpreferably within 1 minute of one another. Therefore, uptake of thenucleic acid via the first indentation may be enhanced by performingsteps 102 and 104 within the predetermined time.

It is further contemplated that the parameters of the first pore (e.g.,size, shape, depth, pattern, etc.) may influence the therapeuticresponse in the infected area. An additional step 108 of applying alight energy may include creating a pore pattern. Suitable pore patternsmay be as simple as one pore or a line of pores. Pore patterns may bemore complex and may include a plurality of pores arranged in lines,continuously curved lines, or a combination thereof. It should beappreciated that pore patterns may influence the uptake of the nucleicinto the plant by providing different dimensional shapes and areas forabsorption. In one embodiment, the method 100 may include an additionalstep 110 of applying the light energy to create a second pore, or anynumber of additional pores.

The method 100 may also include an additional step 106 of applying a waxto the treatment area in an amount effective to seal the first pore andany additional pores. In one embodiment, the step of applying the waxmay be performed after the steps of applying the light energy to thetreatment area and applying the first dosage of a nucleic acid to thetreatment area. In this fashion, the first pore may be sealed from theoutside environment to prevent harmful contaminants from accessing theplant through the first pore. In addition, the wax may reduce the amountof perspiration of the substance to help its absorption into the plant.

FIGS. 2A-2F shows a cross section of a plant 200, which has an infectedsite 206. As discussed above, plant 200 may comprise any type of plantlife, including a tree, vines, forage, perennial crops, row crops, bushcrops, an ornamental plant, annual plants, and grasses. Thus, it shouldbe noted that the methods disclosed herein may be applied to alldifferent plant types to improve the delivery of a nucleic acid into theplant. In one example, plant 200 is a citrus tree.

Infected site 206 may have an area affected by various knowndiseases/pathogens. However, it is contemplated that a plant may have aplurality of infected sites. Contemplated diseases are typicallysystemic in nature, which means that the pathogen occurs within planttissue. Consequently, the treatment of such diseases is difficult tocontrol using conventional surface treatments. For example, contemplateddiseases include HLB, Citrus Tristeza Virus (CTV), Citrus VariegatedChlorosis (CVC), Laurel wilt disease, Fusarium wilt, Phytoplasmas, Zebrachip disease, bacterial kiwifruit vine disease, Chestnut blight, Oakwilt, Fusarium wilt and Pierce's disease.

Infected site 206 may be an area in a plant affected by a disease, suchas HLB, Citrus Tristeza Virus (CTV), Citrus Variegated Chlorosis (CVC)Laurel wilt disease, Fusarium wilt, Phytoplasmas, Zebra chip disease,bacterial kiwifruit vine disease, Chestnut blight, Oak wilt, Fusariumwilt, and Pierce's disease. While FIG. 2A shows infected site 206 withinplant 200, it is contemplated that infected site 206 may reside on theoutside surface of plant 200, such as in the case of citrus canker. Itshould also be noted that while the methods disclosed thus far relate totreating diseased plants, the methods herein may also be applied tohealthy plants for preventive care or to promote overall health (e.g.,fertilizer application).

FIG. 2A shows a light energy 202 may be applied to a treatment area 204of plant 200. Treatment area 204 is typically on a surface of plant 200.However, it is contemplated that treatment area 204 may also be withinplant 200 (i.e., beyond the outer epidermis). Treatment area 204 may bea region of a plant or a specific organ of a plant (e.g., leaf, bark,stem, etc.). In addition, treatment area 204 may comprise a targetedcoverage area of a plant. For example, treatment area 204 may compriseless than 25% of the surface area of the plant (e.g., leaf, stem, trunk,etc.), and more preferably less than 20% of the surface area of a plant.

Treatment area 204 is typically distal from infected site 206. Forexample, treatment area 204 may be a surface of at least one of a leaf,a stem, and a bark, while the infected site is in a phloem of plant 200.In particular, treatment area 204 may be in the dermal system of plant200 while the infected site 206 may be in the vascular system of plant200. Therefore, it is contemplated that the treatment area and theinfected site are in distinct plant systems (e.g., dermis, vascular,ground).

Light energy 202 may be used to create a first pore 208 as shown in FIG.2B. Co-invented U.S. Pat. Nos. 5,660,747 and 5,897,797, and U.S. Pat.Pub. 2005/0226975 describe various techniques for etching the skin ofproduce using energy from a CO₂ laser. It is contemplated that thepresent inventive subject manner may employ some of the techniques inthese references, or modifications thereof, in combination with suitableoperational parameters, to accomplish the objectives of the presentinventive subject matter. For example, light energy 202 may comprise aCO₂ laser that may be configured with suitable parameters (e.g.,size/diameter, quality, spatial intensity distribution, divergence,wavelength, etc.) that are sufficient to produce a desired pore in aparticular treatment surface (e.g., leaf). In some embodiments, suitableparameters for light energy 202 (e.g., a laser) may include a wavelengthhaving about 10 micrometers (e.g., 10.6 micrometers) and a power outputrange between 20-90% at 30-2000 microseconds. It is contemplated thatthe power output of light energy 202 may range from 10-100 watts. Itshould be appreciated that the suitable parameters may be adjusted inreal-time to accommodate various environmental factors that could affectlight energy 202.

As treatment area 204 may comprise different parts of plant 200 (e.g.,leaf and bark), it is contemplated that different parameters of lightenergy 202 may be used for different parts of plant 200. For example,light energy 202 applied to a bark to create a pore typically requiresmore power and dwell than light energy 202 applied to a leaf to create apore. Suitable power settings for applying light energy 202 to a barkmay be in the range of 80-90% power at a dwell 1200-2000 microsecondsdepending on the age of plant 200. On the other hand, grasses wouldtypically require less power in the range of 20-50% power at a dwell of30-120 microseconds.

Other contemplated parameters for light energy 202 include jump speeds,marking speeds, marking intensities, pulse frequencies, and the like.Jump speeds may range from 1 to 3000 mm/s, and more typically between1000-2000 mm/s. Marking speeds may range from 100 to 3000 mm/s, and moretypically from 1000-2000 mm/s. Marking intensity may range from 20-100%,and more typically between 30-70%. Pulse Frequency may range from 15 to20000 Hz, and more typically between 1000-20000 Hz. It should beappreciated that the jump speeds, marking speeds, marking intensities,and pulse frequencies may be applied to all areas of plant 200,including the leaves, branches, stems, bark.

Additionally, it is contemplated that light energy 202 may be generatedusing a 400 mm output lens, which provides a scan window ofapproximately 14 inches by 14 inches without changing the beamcharacteristics and energy. The scan window is the area that may belased by light energy 202. For example, it is contemplated that suchoutput lens provides an optimum focal distance of 15 inches and aneffective depth of focus of 9 inches. It should be appreciated that thefocal point and depth of focus may be changed based on the size of theoutput lens used. For example, a 200 mm lens provides a focal point of 7inches and a depth of focus of 4 inches, and a 800 mm lens provides afocal point of 23 inches and a depth of focus of 15 inches. Thus, itshould be appreciated that a “scribing” laser or continuous wave laseris contemplated, which may be air cooled and designed for the outdoorenvironment.

Furthermore, it should be noted that a galvanometer may be used tocontrol a scribing path of the light energy 102. Typically, thegalvanometer is placed behind the laser output lens, such that lightenergy 102 travels to the galvanometer before the laser output lens.Furthermore, it should be appreciated that more than one galvanometermay be used to control the direction of light energy 102 in twodirections (e.g., x and y directions).

As discussed above, suitable parameters for light energy 202 may varydepending on environmental factors, among other things. To betteroperate a real-time system that adjusts light energy 202, it iscontemplated that a control system (e.g., a feedback loop) havingsensors are integrated with light energy 202. The sensors may be used todetect various parameters affecting the application of light energy 202,such as the different parts of plant 200 (e.g., bark vs. leaf), weather,age of plant 200, depth and size of first pore 208, and the distancefrom the light energy source and treatment area 204. Upon obtainingsensor data on various parameters, the control system may adjust lightenergy 202 to create a pore. For example, a sensor may detect thattreatment area 204 is part of the bark of plant 200 and adjust the powersettings of light energy 202 to create a pore, and at a subsequent pointin time the sensor may detect that treatment area 204 is on the surfaceof grass and adjust the power settings by reducing power of light energy202 to create a pore. In another example, light energy 202 may have adefault setting (e.g., power setting to create a pore on a leaf of plant200) and, upon sensing another part (e.g., stem, bark) of plant 100,control system may adjust light energy 202 to create a pore beforereturning to the default setting. In yet another example, a sensor maybe integrated within light energy 202 to determine whether first pore208 has reached a pre-determined tissue layer.

In contemplated embodiments, light energy 202 may be applied having thelight energy source in contact with treatment area 204. However, inother more typical embodiments, light energy 202 may be applied havingthe light energy source at a distance from treatment area 204. Forexample, the light energy source may be between 4 to 14 inches (e.g., 7inches) from treatment area 204. In another example, light energy 202may comprise a CO₂ laser and treatment area 204 may be on a surface of aleaf whereby the distance between the CO₂ laser and the leaf is between4 to 14 inches, and more preferably, 5 to 8 inches.

First pore 208 may be removed or disrupted portion of treatment area204. In contemplated embodiments, first pore 208 may comprise an openingthrough the treatment area 104. In other words, first pore 208 mayextend through treatment area 204 to expose a surface underneathtreatment area 204. For example, first pore 208 may be a rupture,ablation, or disruption of a cuticle on the leaf. It should beappreciated that first pore 208 is intended to be minimally invasive tothe plant. Therefore, typical diameters for first pore 208 may beapproximately between 100 and 300 μm, and typically have a depth ofapproximately 50 μm. It should be noted that first pore 208 may have agreater or lesser depth, but the depth should generally not exceed 75 μmas it may destroy too many cells.

While first pore 208 may extend through treatment area 204, it is alsocontemplated that first pore 208 may extend partially through treatmentarea 204. In other words, first pore 208 need not be a complete breachthrough treatment area 204 (e.g., the depth of first pore 208 does notextend into the entire thickness of the cuticle). On the other hand, itis also contemplated that first pore 208 may extend through variouslayers past treatment area 204. Preferably, first pore 208 may beconfigured to achieve maximum uptake of the nucleic acid whileminimizing harm to the plant.

Once first pore 308 is created, a first dosage of a nucleic acid 310 maybe applied to treatment area 204 as shown in FIG. 2C. Nucleic acid 210typically promotes the overall health or treatment of plant 200. It iscontemplated that nucleic acid 210 may comprise RNA. Suitable RNA fornucleic acid 210 may include at least one of a RNA interference (RNAi),an antisense RNA (asRNA), a micro RNA (miRNA), a small interfering RNA(siRNA), a double-stranded RNA (dsRNA), a non-coding RNA (ncRNA), andmitochondrial RNA. For example, nucleic acid 210 may be RNAi that atleast one of (i) modifies a gene expression to increase resistance to apathogen in the plant (e.g., interfere with the metabolism ordevelopment process, affect the development of an insect (e.g., citruspsyllid) that feeds on the plant (wings, gut, offspring, etc.)), (ii)suppresses a virus-based vector in the plant, and (iii) modifies a geneexpression to obtain a favorable trait in the plant (e.g., color,flavor, reduced degradation, reduced abiotic stress, etc.). Thus, it iscontemplated that RNAi may be applied via foliar application withenhanced uptake due to the pores created by the light energy.

It is also contemplated that nucleic acid 210 may comprises at least oneof DNA and a nucleic acid analogue. Nucleic acid 210 may also comprise atag for detection to determine whether there was uptake of nucleic acid210 into plant 200. Moreover, nucleic acid 210 may also comprise apharmaceutical tag to enhance or complement the action of the nucleicacid 210. Nucleic acid 210 may have any number of base or base pairs.For example, nucleic acid 110 may have 1-10, 8-20, 12-30, 25-50, 40-100,100-1,000, 1,000-10,000, and 10,000-1,000,000 base pairs.

Nucleic acid 210 may have various suitable structures andcharacteristics. For example, nucleic acid 210 may be single or doublestranded and circular or linear. Nucleic acid 210 may be coding ornoncoding (e.g., noncoding DNA, noncoding RNA). Where protein is encodedby nucleic acid 210, it is contemplated that such proteins are under thecontrol of the regulatory mechanisms specific to plant 210 or a diseaseor pathogen or to neither. Moreover, nucleic acid 210 may comprise atleast one of yeast artificial chromosomes (YACs) and a phagemid plasmid.

Nucleic acid 210 may be with or without sequences that supportreplication. It is also contemplated that the sequences may be fromnon-plants (e.g., animal, bacteria, yeast, fungi, etc.). The sequencesmay also be chimeric (e.g., part animal and part plant, part plant andpart bacteria, etc.).

First dosage of nucleic acid 210 will depend on the type of nucleic acid210 being applied. For example, it is contemplated that nucleic acid 210may have a concentration of 0.1-1 pmol, 1-10 pmol, 10 pmol-1 nmol, 1-10nmol, 10 nmol-1 μmol, 1-10 μmol, 10 μmol-1 mmol, and even higher. Thus,depending on the size of nucleic acid 210 it is contemplated that firstdosage comprises 0.1-1 pmol/ml, 1-10 pmol/ml, 10 pmol/ml-1 nmol/ml, 1-10nmol/ml, 10 nmol/ml-1 μmol/ml, 1-10 μmol/ml, 10 μmol/ml-1 mmol/ml, andeven higher. First dosage of nucleic acid 210 may be buffered ornon-buffered solution.

First dosage of nucleic acid 210 may comprise additional components. Itis contemplated that first dosage of nucleic acid 210 may comprise atleast one of stabilizers, surfactants, and a nucleic acid degradationinhibitor (e.g., DNAse inhibitor, RNAse inhibitor). Thus, first dosageof nucleic acid 210 may be co-administered with other compounds. It iscontemplated that first dosage of nucleic acid 210 may comprise counterions if, for example, at neutral pH.

Nucleic acid 210 may be applied to first pore 208 using various methods,including spraying, dusting, sprinkling, brushing, smearing anddrenching. Application of the first dosage may also be accomplished byintroducing nucleic acid 210 into an irrigation system (e.g., sprinklersystem) that delivers water to plant 200. It should be appreciated thatfirst dosage of nucleic acid 210 may comprise a surfactant to increaseabsorption of nucleic acid 210 in plant 200. Using these applicationtechniques, at least some of nucleic acid 210 enters first pore 208. Itis also contemplated that a more targeted application technique may beused to focus the application of nucleic acid 210 onto the area of firstpore 208.

In yet other embodiments, nucleic acid 210 may produce a harmfulresponse to plant 200 to thereby kill and eliminate plant 200. In thismanner, the methods and devices disclosed herein may be used toselectively remove certain plants from a crop, such as to remove weeds,infected plants, older plants, etc. In such embodiments, first pore 208may be configured to optimize uptake without concern of being minimallyinvasive.

It is further contemplated that nucleic acid 210 is packaged into avariety of lipid soluble nano-particles. These nano-particles shouldprovide for higher retention rates and for improved cuticle penetration.When combined with the use of light energy to create a pore for nucleicacid 210, penetration of nano-particles (or nucleic acids in solution)may be exceptionally enhanced.

Typically, nucleic acid 210 is better absorbed through first pore 208 asshown in FIG. 2D. Among the various reasons for the better absorption,it should be noted that nucleic acid 210 within first pore 208 has amore direct path into plant 200 as opposed to nucleic acid 210 that ison the outer layer (i.e., outer surface of treatment area 204) of plant200. It is preferred that the first dosage of nucleic acid 210 appliedto treatment area 204 is in an amount effective to induce a response(e.g., therapeutic response, biological response) in infected site 206.

Plant 200 may respond in various manners. For example, plant 200 mayhave a therapeutic response or biological that comprises a reduction inthe infected site 206 as shown in FIG. 2E. In another example, thetherapeutic response may comprise at least one of reducing a symptom ofa pathogen, reducing a cause of the disease, and completely curing adisease. It is contemplated that nucleic acid 210 may be RNAi and thetherapeutic response comprises at least one of (i) modifying a geneexpression to increase resistance to a pathogen in plant 200 (e.g.,interfere with the metabolism or development process, affect thedevelopment of an insect (e.g., citrus psyllid) that feeds on the plant(wings, gut, offspring, etc.)), (ii) suppressing a virus-based vector inplant 200, and (iii) modifying a gene expression to obtain a favorabletrait in plant 200 (e.g., color, flavor, reduced degradation, reducedabiotic stress, etc.). Thus, it is contemplated that nucleic acid 210may affect (i) one or more components of plant 200, (ii) one or morecomponents of a symbiotic organism, and (iii) one or more components ofa pathogen.

After creating first pore 208 and applying nucleic acid 210, a wax 212may be applied to treatment area 204 as shown in FIG. 2F. It should beappreciated that wax 212 has several benefits, including preventingpathogen or other harmful contaminants from entering first pore 208,reducing water loss, and reducing perspiration of nucleic acid 210 fromfirst pore 208. Thus, water loss will be mainly localized and should notaffect the overall tree water relations.

FIGS. 3A-3F shows a cross section of a plant 300, which has an infectedsite 306 that is distally located from a treatment area 304 across asystemic pathway 305. For example, treatment area 304 could be locatedon a first leaf and infected site 306 could be located on a second leaf,wherein the first leaf and separate leaf are located on different stemsand/or branches. Light energy 302 may be applied to treatment area 304to create first pore 308 as shown in FIGS. 3A-3B. Once first pore 308 iscreated, a first dosage of nucleic acid 310 may be applied to treatmentarea 304 as shown in FIG. 3C. As discussed above, nucleic acid 310 maybe applied using various application methods and may further comprise asurfactant to increase absorption.

Nucleic acid 310 may absorbed by plant 300 through first pore 308 asshown in FIG. 3D. While nucleic acid 310 has been introduced withinplant 300, it should be noted that in some circumstances the infectedsite 306 is distal from treatment area 304, such that nucleic acid 310is not immediately in contact with infected site 306 and is separated bya systemic pathway. Thus, first dosage of nucleic acid 310 and firstpore 308 may be configured (e.g., the quantity of first dosage, numberof applications of dosage, size and depth of first pore 308, percentageof coverage of first pore 308, etc.) to allow for sufficient uptake anddelivery of the nucleic acid 310 to the infected site 306 to achieve atherapeutic response at the infected site 306.

It is contemplated that nucleic acid 310 may travel through a systemicpathway 305 to arrive at infected site 306 as shown in FIG. 3E. Whilenucleic acid 310 is shown to be present in the area immediately adjacentto first pore 308, it is contemplated that nucleic acid 310 does nothave an effect (e.g., therapeutic or biological response) until ittravels through systemic pathway 305. FIG. 3F shows first pore 308 andnucleic acid 310 may be sufficient to induce a therapeutic response,such as reducing the area of infected site 306. However, it is alsocontemplated that nucleic acid 310 remains localized in the areaadjacent to treatment area 304 (e.g., within the same leaf, or withinthe leaves on a single branch, etc.).

It is contemplated that at least 20%, 30%, 50%, 75%, 90% of nucleic acid310 may be absorbed by at least one of the phloem and the xylem in plant300. In some embodiments, nucleic acid 310 may be localized in thephloem adjacent to treatment area 304 and not travel to the roots.However, it is also contemplated that nucleic acid 310 may be absorbedby the phloem and/or xylem to thereby travel through the vascular systemof plant 300. Regardless of the mode of transport, it should be notedthat limited application of nucleic acid 310 may generate an effectthroughout 1-5%, 5-15%, 15-35%, 35-60%, 60-90%, and 90-100% of the plantwithin 1 hour, 2 hours, 5 hours, 10 hours, 18 hours, 1 day, or 2 days.

As discussed above, a treatment area may have more than one pore and maycomprise a pore pattern. FIGS. 4A-4D show a treatment area 404 that mayhave various pore patterns 403 a-d. For example, pattern 403 a is asingle solid line, pattern 403 b comprises multiple solid lines, pattern404 c comprises a plurality of circular dots equally distanced from oneanother, and pattern 404 d comprises a plurality of randomly spaceddots. However, while creating a plurality of pores provides increasedsites for direct introduction of the substance into the plant, it isgenerally preferred that not more than 25% of the surface area of theplant be covered with pores to avoid permanent damage to the plant. Insome applications, more than 25% of the surface area of the plant maycomprise pores, but it is generally recommended that an additional stepof waxing is applied on the pores to protect the plant from harmfulsubstances.

It should be recognized that a treatment area may span to various partsof a plant. For example, treatment area 404 may encompass the leaves,stems and the trunk of a plant. In another example, treatment area 404may be limited to just one region or part of the plant (e.g., leaves,stems, bark, roots, etc.). Thus, it is contemplated that light energymay be applied to a first site and a second site on a plant that aredifferent in kind (e.g., different tissues, cells, organs, systems ofplant, distal).

It should be appreciated that the pores may vary in size and shape toaccount for differences in the absorption rate between various parts ofthe treatment area (e.g., the bark and the cuticle). For example, if itis determined that nucleic acids on a pore on the bark are absorbedslower than nucleic acids on a pore on the cuticle, then the pore on thebark may be larger to compensate for the slower absorption rate.Moreover, pores may be created to promote capillary action of thenucleic acids.

In another method, light energy may be used to create a first porepattern. A first dosage of nucleic acid may be applied to the first porepattern and a second dosage of nucleic acid may be applied to the firstpore pattern after the first dosage is applied. For example, a firstdosage of the first nucleic acid may be applied, then after apre-determined time period, a second dosage of the first nucleic acidmay be applied. In another example, a first dosage of the first nucleicacid may be applied, and then a first dosage of a second nucleic acidmay be applied after a pre-determined time period.

The second nucleic acid may be any of the various nucleic acidsdescribed above. Additionally, the second substance may further includea booster or a catalyst for the first nucleic acid to further activatethe first nucleic acid. It is contemplated that the second nucleic acidmay produce a second biological response, wherein the second biologicalresponse may be connected to the first biological response or distinctfrom the first biological response. It is contemplated that the firstand second dosage amounts are equal. In other embodiments, the first andsecond dosage amounts vary depending on the effective dosage amountrequired to produce a biological response.

Thus, it discovered that nucleic acids may be effectively delivered to aplant using light energy. The nucleic acids may be effective to interactwith pathogens in the plant. However, it should be appreciated thateffective delivery of nucleic acids may also interact with plantmetabolism. Therefore, the delivery methods described herein may also beused to regulate genes within the plant. Due to the ability to modifygenetic activity of the plant, nucleic acids applied to the plant mayinteract with nucleic acids that are normally found in the plant or withnucleic acids that are not normally found in a plant (e.g., transgenes).It is contemplated that nucleic acids may be delivered to a plant toactivate or deactivate transgenes, which may include nucleic acidspreviously introduced into the plant.

FIGS. 5A-5B show an embodiment of an apparatus 500 configured fordelivering at least one nucleic acid to a plant 502. The apparatus maybe mounted on a movable platform 504 for ease of use. The basic unit maycomprise an electronically controlled robotic arm 506 containing: (i) alaser light emitter 508 to disperse a light to a treatment area orsecond site on a plant 502; (ii) a nucleic acid nozzle applicator 510;and (iii) a wax applicator 512 to seal the treated area as shown in FIG.5A. The laser light emitter 508, nucleic acid nozzle applicator 510, andwax applicator 512 may be nozzles. All these nozzles may be mounted atthe end of the arm on a movable bracket. In sequence, the laser lightemitter 508 may emit light energy to create a momentary breach in thetreatment area or second site on the plant 502, the nucleic acid nozzleapplicator 510 may deliver the nucleic acid over the treatment area orsecond site on the plant 502, and the wax applicator 512 may reseal thetreatment area or second site on the plant as the system moves forward.The system design may reduce the amount of substance needed by applyingit only over the treatment area or second site on the plant, and notover the entire canopy/area and also ensures that the pores may besealed to protect against water loss and other pathogens.

The apparatus 500 may further comprise five individual arms 520, 522,524, 526, 528 as shown in FIG. 5B, each independently controlled andelectronically actuated. These arms may be stacked vertically, with eachlaser having a scan window of approximately 14″×14″. This will give amaximum laser treated height of approximately 6 ft. The design of thesystem may be modular so that more laser arms may be added vertically toaccommodate larger trees if needed. The entire apparatus 500 may bemounted on the movable platform 504 which will be pulled through thegrove by a tractor 540 at 2.3-2.7 mph. As the apparatus moves down thegrove, optics will guide each arm to determined positions on the canopy,avoiding entanglement with branches of uneven length.

Each independently controlled arm 520, 522, 524, 526, 528, with thelaser system and attached applicator nozzle assembly, may be designed totypically move in the horizontal direction. However, each laser systemand assembly arm may move independently from the other arms. Thisindependent scheme may allow each laser system to follow the contour ofthe tree canopy in its scan path. Multiple sensors on the articulatingarm and laser system may ensure that it traces the tree canopy. Theentire laser system may also be raised or lowered vertically dependingon tree height. In addition, because of tree canopy irregularities anddiffering tree height, sensors on the laser may turn it “off and on”based on the presence of canopy to be treated within its scan path. Thelaser system and applicator nozzles may be attached on the arm using arotating mount (not shown). This rotating mount may allow the lasers tobe pointed at an angle to the tree canopy to better laser the adaxial(top) of the leaves.

In such a harsh working environment, the platform 504, on which theentire laser system is mounted on, may have an isolation design so thatit will eliminate (as much as possible) the transfer of motion from theplatform 504 to the apparatus 500 as it moves through the grove. Thehorizontal articulating robotic arms may also have isolation mounts 530that are designed to minimize any further vibrations from affecting theoperation of the laser system in the field. A generator 532 and tankswith pumps 534 may be mounted on the platform 504, as shown in FIG. 5A.The generator 532 provides sufficient power for the laser systems, pumpsand associated electronic equipment. The tanks provide the storage forat least one of the substance and wax. The two pumps deliver thesubstance and wax to the applicator nozzles. The overall system designpreferably takes into account environmental working conditions, heat,humidity, rain and varying grove conditions.

To the best of the inventor's knowledge, this is the first time RNAi hasbeen introduced to the phloem after laser poration. Unexpectedly, theefficiency of RNAi was very high. Typically, at least 50%, 60%, 75%,90%, and even 100% of all treatment areas resulted in a therapeuticresponse. Viewed from a different perspective, the inventor discoveredthat RNAi may be introduced into a plant by administering RNAi directlyto the phloem through pores created by a laser.

The previous description of the disclosed embodiments is provided toenable any person skilled in the art to make or use the presentdisclosure. Various modifications to these embodiments will be readilyapparent to those skilled in the art, and the generic principles definedherein may be applied to other embodiments without departing from thespirit or scope of the disclosure. Thus, the present disclosure is notintended to be limited to the embodiments shown herein but is to beaccorded the widest scope consistent with the principles and novelfeatures disclosed herein.

Unless otherwise expressly stated, it is in no way intended that anymethod set forth herein be construed as requiring that its steps beperformed in a specific order. Accordingly, where a method claim doesnot actually recite an order to be followed by its steps or it is nototherwise specifically stated in the claims or descriptions that thesteps are to be limited to a specific order, it is in no way intendedthat an order be inferred, in any respect. This holds for any possiblenon-express basis for interpretation, including: matters of logic withrespect to arrangement of steps or operational flow; plain meaningderived from grammatical organization or punctuation; the number or typeof embodiments described in the specification.

It will be apparent to those skilled in the art that variousmodifications and variations may be made without departing from thescope or spirit. Other embodiments will be apparent to those skilled inthe art from consideration of the specification and practice disclosedherein. It is intended that the specification and examples be consideredas exemplary only, with a true scope and spirit being indicated by thefollowing claims.

What is claimed is:
 1. A method of delivering at least one nucleic acid to a plant having an infected site, comprising: applying a light energy to a surface of the plant to create a pore; applying at least one nucleic acid to the pore in an amount effective to induce a therapeutic response in the infected site; and wherein the infected site is in a phloem of the plant.
 2. The method of claim 1, wherein the at least one nucleic acid comprises RNA.
 3. The method of claim 2, wherein the at least one nucleic acid comprises at least one of a RNA interference (RNAi), an antisense RNA (asRNA), a micro RNA (miRNA), a small interfering RNA (siRNA), a double-stranded RNA (dsRNA), a non-coding RNA (ncRNA), mitochondrial RNA (mtDNA), and combinations thereof.
 4. The method of claim 3, wherein the plant comprises a tree, a vine, a forage, a perennial crop, a row crop, a bush crop, an ornamental plant, an annual plant, or a grass.
 5. The method of claim 1, wherein the at least one nucleic acid comprises DNA.
 6. The method of claim 1, further comprising applying a second nucleic acid to the pore.
 7. A method of modifying a plant having an infected site, comprising: applying a light energy to a surface of the plant to create a pore; applying at least one nucleic acid to the pore to allow uptake and delivery to the infected site; wherein the at least one nucleic acid is applied in an amount effective to produce a response; and wherein the infected site is in a phloem of the plant.
 8. The method of claim 7, wherein the at least one nucleic acid comprises RNA.
 9. The method of claim 8, wherein the at least one nucleic acid comprises at least one of an RNA interference (RNAi), an antisense RNA (asRNA), a micro RNA (miRNA), a small interfering RNA (siRNA), a double-stranded RNA (dsRNA), a non-coding RNA (ncRNA), mitochondrial RNA, and combinations thereof.
 10. The method of claim 9, wherein the plant comprises a tree, a vine, a forage, a perennial crop, a row crop, a bush crop, an ornamental plant, an annual plant, or a grass.
 11. The method of claim 7, wherein the at least one nucleic acid comprises DNA.
 12. The method of claim 7, further comprising applying a second nucleic acid to the pore.
 13. The method of claim 7, wherein the response comprises at least one of (i) modifying a gene expression to increase resistance to a pathogen in the plant, (ii) suppressing a virus-based vector in the plant, and (iii) modifying a gene expression to obtain a favorable trait in the plant.
 14. A method of delivering at least one nucleic acid to a plant having an infected site, comprising: applying a light energy to a treatment area on the plant to create a pore; applying at least one nucleic acid to the pore in an amount effective to reduce a symptom of a pathogen in the infected site; and wherein the infected site is in a phloem of the plant.
 15. The method of claim 14, wherein the applying a light energy and the applying a first dosage is within a predetermined time.
 16. The method of claim 14, wherein the treatment area is a surface of at least one of a leaf, a stem, and a bark.
 17. The method of claim 14, wherein the applying a light energy further comprises creating a second pore.
 18. The method of claim 14, wherein the at least one nucleic acid comprises RNA.
 19. The method of claim 18, wherein the at least one nucleic acid comprises at least one of a RNA interference (RNAi), an antisense RNA (asRNA), a micro RNA (miRNA), a small interfering RNA (siRNA), a double-stranded RNA (dsRNA), a non-coding RNA (ncRNA), mitochondrial RNA (mtDNA), and combinations thereof.
 20. The method of claim 19, wherein the plant comprises a tree, a vine, a forage, a perennial crop, a row crop, a bush crop, an ornamental plant, an annual plant, or a grass.
 21. The method of claim 14, wherein the at least one nucleic acid comprises DNA. 